The invention disclosed in this patent document relates to transmembrane receptors, and more particularly to G protein-coupled receptors for which the endogenous ligand has been identified (xe2x80x9cknown GPCRxe2x80x9d), and specifically to known GPCRs that have been altered to establish or enhance constitutive activity of the receptor. Most preferably, the altered GPCRs are used for the direct identification of candidate compounds as receptor agonists, inverse agonists or partial agonists for use as therapeutic agents.
Although a number of receptor classes exist in humans, by far the most abundant and therapeutically relevant is represented by the G protein-coupled receptor (GPCR or GPCRs) class. It is estimated that there are some 100,000 genes within the human genome, and of these, approximately 2%, or 2,000 genes, are estimated to code for GPCRs. Receptors, including GPCRs, for which the endogenous ligand has been identified are referred to as xe2x80x9cknownxe2x80x9d receptors, while receptors for which the endogenous ligand has not been identified are referred to as xe2x80x9corphanxe2x80x9d receptors. GPCRs represent an important area for the development of pharmaceutical products: from approximately 20 of the 100 known GPCRs, 60% of all prescription pharmaceuticals have been developed.
GPCRs share a common structural motif. (All these receptors have seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane (each span is identified by number, i.e., transmembrane-1 (TM-1), transmembrane-2 (TM-2), etc.). The transmembrane helices are joined by strands of amino acids between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the exterior, or xe2x80x9cextracellularxe2x80x9d side, of the cell membrane (these are referred to as xe2x80x9cextracellularxe2x80x9d regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively). The transmembrane helices are also joined by strands of amino acids between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the interior, or xe2x80x9cintracellularxe2x80x9d side, of the cell membrane (these are referred to as xe2x80x9cintracellularxe2x80x9d regions 1, 2 and 3 (IC-1, IC-2 and IC-3), respectively). The xe2x80x9ccarboxyxe2x80x9d (xe2x80x9cCxe2x80x9d) terminus of the receptor lies in the intracellular space within the cell, and the xe2x80x9caminoxe2x80x9d (xe2x80x9cNxe2x80x9d) terminus of the receptor lies in the extracellular space outside of the cell.
Generally, when an endogenous ligand binds with the receptor (often referred to as xe2x80x9cactivationxe2x80x9d of the receptor), there is a change in the conformation of the intracellular region that allows for coupling between the intracellular region and an intracellular xe2x80x9cG-protein.xe2x80x9d It has been reported that GPCRs are xe2x80x9cpromiscuousxe2x80x9d with respect to G proteins, i.e., that a GPCR can interact with more than one G protein. See, Kenakin, T., 43 Life Sciences 1095 (1988). Although other G proteins exist, currently, Gq, Gs, Gi, Gz and Go are G proteins that have been identified. Endogenous ligand-activated GPCR coupling with the G-protein begins a signaling cascade process (referred to as xe2x80x9csignal transductionxe2x80x9d). Under normal conditions, signal transduction ultimately results in cellular activation or cellular inhibition. It is thought that the IC-3 loop as well as the carboxy terminus of the receptor interact with the G protein.
Under physiological conditions, GPCRs exist in the cell membrane in equilibrium between two different conformations: an xe2x80x9cinactivexe2x80x9d state and an xe2x80x9cactivexe2x80x9d state. A receptor in an inactive state is unable to link to the intracellular signaling transduction pathway to produce a biological response. Changing the receptor conformation to the active state allows linkage to the transduction pathway (via the G-protein) and produces a biological response.
A receptor may be stabilized in an active state by an endogenous ligand or a compound such as a drug. Recent discoveries, including but not exclusively limited to modifications to the amino acid sequence of the receptor, provide means other than endogenous ligands or drugs to promote and stabilize the receptor in the active state conformation. These means effectively stabilize the receptor in an active state by simulating the effect of an endogenous ligand binding to the receptor. Stabilization by such ligand-independent means is termed xe2x80x9cconstitutive receptor activation.xe2x80x9d
Traditional ligand-dependent screens seek to indirectly identify compounds that antagonize the action of the ligand on the receptor in an effort to prevent ligand-induced activation of the receptor. However, such compounds, sometimes referred to as neutral-antagonists, generally would not be expected to affect the ligand-independent activity, or overactivity, of the receptor and the subsequent abnormal cellular response that can result from this overactivity. This is particularly relevant to a growing number of diseases, such as those identified in the table below, that have been linked to overactive GPCRs, because traditional neutral-antagonists will not block the abnormal ligand-independent activity of these receptors.
Disclosed herein are non-endogenous versions of endogenous, known GPCRs and uses thereof.